Human embryonic stem (hES) cells are being studied as potential source of cells for the treatment for many diseases (e.g. diabetes, spinal cord injury, Parkinson's, leukemia, congestive heart failure, etc.). These same cells are also being touted as the ideal cell source for ex vivo tissue engineering or in situ regenerative medicine. The successful integration of hESC into such therapies will hinge upon three critical steps: 1) stem cell expansion in number without differentiation (i.e., self-renewal); 2) differentiation into a specific cell type or collection of cell types; and, 3) promotion of their functional integration into existing tissue. Precisely controlling each of these steps will be essential to maximize hES cell's therapeutic efficacy. However, it is difficult to precisely control the behavior of hES cells, since environmental conditions for self-renewal and differentiation are incompletely understood. Historically, hES cells have typically been grown in monolayer culture with a feeder layer of mouse cells (i.e., irradiated but viable cells) and/or conditioned with media derived from these cells. These methods increase the risk of zoonoses acquired from the murine feeder cells and culture medium, and have significant disadvantages in reproducibility and scalability that greatly limit their clinical potential. To date, no culture conditions have been identified that would be suitable for hES cell production at the scale required to treat a common disease such as diabetes or congestive heart failure, or production of tissue equivalents ex vivo. We propose to develop two platform technologies presenting a tunable completely synthetic extracellular matrix and chemically-defined media to control the self- renewal/expansion of hESCs. If hES cells can be derived and maintained within a completely synthetic environment, then it will be possible to eliminate pathogen transmission associated with animal-derived materials, provide a scalable basis for large-scale production of hESCs, and provide a precise base for further development to control hES cell differentiation. Furthermore, a significant result of this application will be a technology platform that can be generally applied to numerous stem cell populations and used to investigate the basic biological/developmental mechanisms underlying self-renewal, drug and chemotherapy screening, and ultimately directed differentiation. The following specific aims are proposed. Specific Aim 1 to develop and characterize nanopatterned cell culture substrata where the size, peptide ligand density, number/cell body, and spatial arrangement of integrin-engaging domains will be varied to control cell and colony morphology. Specific Aim 2 to develop a synthetic culture system, termed variable moduli interpenetrating polymer networks, with tunable ligand presentation (i.e., peptide type, ligand density, geometry) and material moduli. Specific Aim 3 to evaluate the platforms developed in Aims 1 & 2 to support the long-term growth (5-10 passages) of human ES cells in chemically-defined media. Public Health Relevance Statement (provided by applicant): This application will focus specifically on engineering a tunable and well-defined environment presenting hESCs with a completely synthetic extracellular matrix and chemically-defined medium to promote self renewal. The result will be a synthetic microenvironment that can both serve as a regenerative medicine technology platform for large scale hESC expansion, as well as provide a novel and highly modular system for dissecting basic signaling mechanisms underlying hESC self-renewal.